Altered methylation of glucosylceramide synthase promoter regulates its expression and associates with acquired multidrug resistance in invasive ductal breast cancer

// Jiannan Liu 1, * , Xiaofang Zhang 2, * , Aina Liu 1 , Daoping Zhang 3 , Yi Su 1 , Ying Liu 1 , Dong You 1 , Leilei Yuan 4 , Xiangshuo Kong 1 , Xiaodan Wang 1 , Ping Sun 1 1 Department of Oncology, Yuhuangding Hospital, Yantai, Shandong, 264000, P. R. China 2 Department of Pathology, Shandong University School of Medicine, Jinan, Shandong, 250012, P. R. China 3 Department of Rehabilitation, Qianfoshan Hospital, Jinan, Shandong, 250014, P. R. China 4 Department of Radiology, Taian Central Hospital, Taian, Shandong, 271000, P. R. China * These authors are contributed equally to this work Correspondence to: Xiaofang Zhang, email: xiaofangzhang@sdu.edu.cn Ping Sun, email: sunping20039@hotmail.com Keywords: glucosylceramide synthase, DNA methylation, 5-Aza-dc, breast cancer Received: October 17, 2015      Accepted: April 16, 2016      Published: May 13, 2016 ABSTRACT Overexpression of glucosylceramide synthase ( GCS ) increases multidrug resistance (MDR) in many cancer cells. However, its mechanism is unknown. The aim of the present study is to detect the association of methylation at the GCS gene promoter with its expression and MDR in invasive ductal breast cancer. 40 cases GCS -positive and 40 cases GCS -negative primary breast carcinoma samples, three drug-sensitive breast cancer cell lines and one multidrug-resistant breast cancer cell line were used. Immunohistochemistry, methylation-specific PCR (MSP), quantitative real-time (qPCR), westernblot and cytotoxicity assay techniques were employed. Thwe results revealed that there was a statistically negative correlation between GCS CpG islands methylation and GCS phenotype in patients with breast cancer. GCS CpG islands methylation was negatively associated with high ER, meanwhile positively with high HER-2 status. Similar results were obtained from the analysis of breast cancer cell lines. Treatment with the demethylating agent 5-aza-2′-deoxycytidine (5-Aza-dc) changed the GCS promoter methylation pattern in three sensitive cells and also caused increased drug resistance of them. These results suggested that the changes of DNA methylation status of the GCS promoter correlates with multidrug resistance in breast cancer.